Contribution of NKT cells to the immune response and pathogenesis triggered by respiratory viruses

Abstract

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) cause acute respiratory tract infections in children worldwide. Natural killer T (NKT) cells are unconventional T lymphocytes, and their TCRs recognize glycolipids bound to the MHC-I-like molecule, CD1d. These cells modulate the inflammatory response in viral infections. Here, we evaluated the contribution of NKT cells in both hRSV and hMPV infections. A significant decrease in the number of neutrophils, eosinophils, and CD103⁺DCs infiltrating to the lungs, as well as an increased production of IFN-γ, were observed upon hRSV-infection in CD1d-deficient BALB/c mice, as compared to wild-type control mice. However, this effect was not observed in the CD1d-deficient BALB/c group, upon infection with hMPV. Importantly, reduced expression of CD1d in CD11b⁺ DCs and epithelial cells was found in hRSV -but not hMPV-infected mice. Besides, a reduction in the expression of CD1d in alveolar macrophages of lungs from hRSV- and hMPV-infected mice was found. Such reduction of CD1d expression interfered with NKT cells activation, and consequently IL-2 secretion, as characterized by in vitro experiments for both hRSV and hMPV infections. Furthermore, increased numbers of NKT cells recruited to the lungs in response to hRSV- but not hMPV-infection was detected, resulting in a reduction in the expression of IFN-γ and IL-2 by these cells. In conclusion, both hRSV and hMPV might be differently impairing NKT cells function and contributing to the immune response triggered by these viruses.

Keywords: Human respiratory syncytial virus; human metapneumovirus; natural killer T cells; pulmonary inflammation; viral infection.

Figure 1.

Expression levels of CD1d in infiltrated cells into the lungs from hRSV- and hMPV-infected mice and IL-2 secretion by NKT cells hybridoma in vitro. Wild-type [WT] BALB/c mice were infected with 1 × 10⁶ PFU of hRSV (a) or hMPV (b) and 3-days post-infection lungs were removed, homogenized and cells were stained to detect the expression of CD1d in alveolar macrophages (CD45⁺CD11b⁻CD11c⁺CD64⁺CD24), CD103⁺ DCs (CD45⁺CD11b⁻CD11c⁺CD64⁻CD24⁺), monocytes (CD45⁺CD11b⁺MHCII⁻CD64^−/low), epithelial cells (CD45⁻Epcam⁺), and DCs CD11b⁺ (CD45⁺CD11b⁺CD11c⁺MHCII⁺CD24⁻CD64⁻). The mean fluorescence intensity (MFI) for CD1d on these cells was analyzed by flow cytometry. Combined data from two independent experiments (n = 4 to 5 mice per group and independent experiment) are shown. Kruskal–Wallis test and Mann–Whitney U tests were performed to assess statistical differences. * P < 0.05 ** P < 0.01 *** P < 0.001. Bars represent mean ± SEM. Pre-pulsed α-GalCer and hRSV- (c) or hMPV- (d) infected DCs from wild-type C57BL/6 mice were co-cultured with iNKT cells hybridoma and IL-2 production was evaluated by ELISA. UT: Untreated; VH: Vehicle; α-GAL: α-Galcer. Combined data from three independent experiments (n = 2–3 treatment per group and experiment) are shown. Kruskal–Wallis and Mann–Whitney U test were performed to assess statistical differences. * P < 0.05. Lines represent mean ± SEM.

Figure 2.

Infection with hRSV induces lower neutrophils, eosinophils, and CD103⁺ DCs infiltration into the lungs in CD1d-deficient mice. Wild-type [WT] and CD1d-deficient (CD1d^-/-) BALB/c mice were infected with 1 × 10⁶ PFU of hRSV and 3 days after infection, BALF, and lungs were obtained. Bodyweight was measured for 3 days (a). Neutrophils (b), eosinophils (c), and CD103⁺DCs (d) in the BALF were evaluated by flow cytometry. Lung damage was evaluated by lung histology (e) and clinical score (f). Viral loads were evaluated by RT-qPCR (g). Combined data from three independent experiments (n = 2 to 3 mice per group and independent experiment) are shown. Kruskal-Wallis test and Mann–Whitney U test were performed to assess statistical differences. * P < 0.05. Bars represent mean ± SEM.

Figure 3.

No significant changes were detected during an hMPV-infection between wild-type and CD1d-deficient BALB/c mice. Wild-type [WT] and CD1d-deficient BALB/c (CD1d^-/-) mice were infected with 1 × 10⁶ PFU of hMPV and 3 days after infection BALF and lungs were obtained. Bodyweight was measured for 3 days (a). Neutrophils (b), eosinophils (c), and CD103⁺DCs (d) in the BALF were evaluated by flow cytometry. Lung damage was evaluated by lung histology (e) and clinical score (f). Viral loads were evaluated by RT-qPCR (g). Combined data from three independent experiments (n = 2 to 3 mice per group and independent experiment) are shown. Kruskal–Wallis test and Mann–Whitney U test were performed to assess statistical differences. * P < 0.05. Bars represent mean ± SEM.

Figure 4.

hRSV- and hMPV-mediated NKT activation induce lower cytokines levels than mock-treated mice in the lungs. Wild-type [WT] BALB/c mice were intranasally infected with 1 × 10⁶ PFU of hRSV or hMPV -or mock inoculated- and 3 days after infection, lungs were removed. Percentage of NKT cells in the lungs of hRSV (a) and hMPV (b) infected mice was assessed. Then, lungs cells were treated for the induction of the secretion of cytokines and intracellular staining of IFN-γ, IL-2, and IL-4 of NKT was performed (c) (d).Combined data from three independent experiments (n = 4–5 mice per group) are shown. Kruskal–Wallis test and Mann–Whitney U test were performed in order to assess statistical differences. * p < 0.05, ** p < 0.001. Bars represent mean ± SEM.

Figure 5.

Cytokine profiling in BALF and CD8⁺ T cells population in mice infected with hRSV or hMPV. Wild-type [WT] and CD1d-deficient (CD1d^-/-) BALB/c mice were infected with 1 × 10⁶ PFU of either hRSV or hMPV and 3 days after infection BALF was obtained. IFN-γ, IL-5, IL-6, IL-1β, and IL-10 were measured in those samples from hRSV- (a-e) and hMPV-infected (g-k) mice. CD8⁺ T cells population in spleen from hRSV- (f) and hMPV-infected (l) mice were measured. Combined data from three independent experiments (n = 2 to 3 mice per group and experiment) are shown. Kruskal–Wallis test and Mann–Whitney U test were performed in order to assess statistical differences. * P < 0.05. Bars represent mean ± SEM.